Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
Reports on the ability of stem cells to regenerate bodily tissues and on unique plasticity of the cellular stem cell compartment have opened the possibility of targeted tissue repair. Hematopoietic stem cells (HSCs) exhibit both a capacity for self-renewal and the potential to generate into a variety of specialised blood cell types (lineages) upon exposure to specific microenvironments. The process of differentiation of different blood cells, ie hematopoiesis, provides a valuable model for examining how genetic programs are established and executed in vertebrates, and also how homeostasis of blood formation is altered in leukemias. Recent findings not only indicate how this may be achieved but also show the extraordinary plasticity of tissue stem cells in vivo.
Hematopoiesis is usually depicted in a hierarchical fashion, with HSCs giving rise first to progenitors and then to precursors with varying capacities to differentiate down multiple or single lineage pathways. Studies investigating the plasticity of cells committed to a particular lineage have found that the process of lineage switching required transformation and/or expression of oncogenes. Moreover, when evidence for lineage switching was observed it was found to have occurred only in small percentages of cells (Klinken et al, Cell 53: 857-867, 1988; Graf et at, Blood 99: 3089-3101, 2002).
Specifically, experiments with Abelson virus transformed pre-B lymphoma cell line treated with 5-azacystidine resulted in a subset of cells exhibiting macrophage-like properties, including the ability to adhere to plastic as well as phagocytic and esterase activities (Boyd et al, Nature 297:691-693, 1982). More recent experiments have demonstrated that concomitant expression of E-myc and ν-raf oncogenes in B lymphoma cell lines or pre-leukemic bone marrow B lineage cells can lead to their conversion to macrophages. These transfected macrophage-like cells processed multiple myelomonocytic markers such as colony stimulating receptor-1 and expressed lysozyme but also retained features of the original cells such as immunoglobulin rearrangement. In addition to transition to macrophages, B-lineage cells were also reported to transit to granulocytes upon transfection with max gene (Lindeman et al, Immunity 1: 517-527, 1994). Significantly, only very small fractions of cells expressing trans-genes actually switched lineage, indicating that the transition from one lineage to another is a complex process which is not completely understood (Klinken et al, Cell 53: 857-867, 1988; Borzello et al, Mol. Cell. Biol. 10:2703-2714, 1990).
An alternate experimental approach which investigated hematopoietic lineage plasticity involved the generation of gene-knockout mice. Specifically, B lineage cells derived from Pax-5 knockout mice exhibited characteristics of pre-B cells such as expression of B220, AA4.1, SL chain and c-Kit (low) but not CD19. These cells also exhibited characteristics of pluripotent stem cells such as continuous self-renewal on stromal cells in presence of interleukin 7 (IL-7), horning to the bone marrow after transplantation, and the ability to differentiate into most hematopoietic cell types both in vivo and in vitro. For example, when transplanted into RAG2 knockout mice these cells differentiate into T cells (Rolink et al, Nature 401:556-562, 1999). When grown in the presence of different cytokines or growth factors they differentiated into a range of different cell types. For example, treatment with IL-2 resulted in conversion to natural killer cells (NK cells), treatment with CSF-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF), resulted in conversion to dendritic cells, treatment with CSF-1 resulted in conversion to macrophages, and treatment with IL-3, IL-6, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) resulted in conversion to neutrophils (Nutt et al, Nature 401: 556-562, 1999).
Although plasticity appeared to be demonstrated with cells derived from Pax-5 knockout mice, cells exhibiting a phenotype comparable to the Pax-5 knockout pre-B cells most likely do not exist in wild type animals as culture of pre-B cells from wild type mice is problematic (Kee et al, Curr. Opin. Immunol. 13: 180-185, 2001).
Other studies using transgenic mice investigating plasticity of common lymphoid progenitors (CLPs), showed that CPLs from a transgenic mouse line ectopically expressing human IL-2 receptor β chain when cultured on stromal cells in presence of IL-7 differentiated into B cells and NK cells. When IL-2 was added to the culture they formed granulocytes and macrophages. However, these results could not be repeated from control CLPs grown under the same conditions (Kondo et al, Nature 407: 383-386, 2000). Accordingly, the results of these studies seem to suggest that inducing cell plasticity requires the expression of genes not normally expressed by committed cells.
The cells exhibiting phenotypic plasticity detailed in the experiments mentioned above were either derived from mouse lymphomas, were cells transformed by an oncogene, or altered by mutations. Limited data exist on the plasticity of normal B-cells. One set of experiments described the conversion of CD19, DJ-rearrangement positive and B220 negative B cells from bone marrow of adult mice into adherent macrophage like cells when cultured with IL-3, IL-6 and GM-CF. However, conversion into NK or T cells was not possible to achieve (Montecino-Rodriguez et al, Nat. Immunol. 2: 83-88, 2001).
Very limited data is available on the reverse transformation from myeloid lineage into B lymphoid lineage. The experiments that have been undertaken were primarily concerned with forced expression of early B lineage specific transcription factors encoded by E2A, EBF and RAG gene. For example, forced expression of E2A caused the cells to lose their adherent properties, down-regulate the expression of Mac-1 and c-fms, and induce a number of B lineage specific genes including an ability to form κ chain in response to mitogens (Kee et al, J. Exp. Med. 188: 699-713, 1998). The fact that these cells did not acquire full B-lineage phenotype suggests that the combination of E2A, EBF, and RAG gene expressions is not sufficient for lineage specification and that additional genes must be involved (Romanow et al, Mol. Cell. 5: 343-353, 2000).
There are also examples of the possible conversion between T-lineage and macrophages. A subset of purified pro-T cells when cultured in the conditioned medium from a thymic stromal cell line generated functional macrophages. The same conversion was observed in the presence of a combination of IL-6, IL-7 and macrophage colony-stimulating factor (M-CSF; CSF-1), but at a much lower frequency (Lee et al, J. Immunol. 166: 5964-5989, 2001).
The most described transdifferentiation is arguably the plasticity within myeloid-erythroid compartment. Most experiments in this field are based on enforced transcription factor expression. The most reproducible and predictable experimental system allowing direct differentiation of cultured cell lines from one lineage to another is based on transformation of avian erythroid-megakaryocytic progenitors by the Myb-Ets-encoding leukemia virus. These E26-MEP cells express a number of megakaryocytic and stem cell surface markers such as MEP21/thrombomucin/PCLP1 and GPIIaIIb/CD41 (Graf et al, Cell 70:201-213, 1992; McNagny et al, J. Cell. Biol. 138:1395-14-7, 1997) as well as GATA-1 and FOG-1 but no myelomonocytic cell surface markers and no or low levels of PU.1, C/EBPα and C/EBPβ. If ν-Ets is inactivated in these cells, they differentiate into erythroid cells (Golay et al, Cell 55: 1147-1158, 1987; Rossi et al, Curr. Biol. 6: 866-872, 1988). Alternatively, inactivation of Myb leads to conversion of these cells into thrombocytes (Frampton et al, EMBO J. 14: 2866-2875, 1995). Through an introduction of oncogenes of the ras pathways via retroviral transformation or by activating protein kinase C (PKC) the cells can be committed to becoming either eosinophils or myeloblasts, depending on the strength of the signal (Graf et al, Cell 70:201-213, 1992; Rossi et al, EMBO J. 15: 1894-1901, 1996). It is widely acknowledged that maintenance of multi-potent or differentiated state is the result of an on-going process and that activation or repression of a single (or a few) nuclear regulators may lead to differentiation, lineage switching or de-differentiation (Orkin, Nature Rev. Genet. 1: 57-64, 2000). In this context, study of nuclear transcription factors that are restricted in their expression to particular lineages is of particular interest, as they establish gene expression programmes intrinsic to cell diversification. Whilst growth factors are important in sustaining hematopoiesis, cell viability and proliferation, they are not necessarily instructive for pathway differentiation (Sokolovsky et al, Proc. Natl. Acad. Sci. USA 95: 6573-6575, 1998; Stoffel et al, Proc Natl. Acad. Sci. USA 96: 698-702, 1999).
The complexity of lineage differentiation has been further illustrated by studies which indicate that the concentration or level at which a given factor is expressed may influence direction of lineage differentiation. Specifically, in a transformed chicken progenitor system (Kulessa et al, Genes Dev. 9: 1250-1262, 1995), the lineage outcome correlated with the level of GATA-1 expression. At low levels, it generated eosinophils and at high levels it produced erythroid and megakaryocytic cells. Recently, it has also been found that high levels of PU.1 favour the development of macrophages, whereas at lower levels, B cells are generated (DeKoter et al, Science 288: 146-149, 2000).
It has also recently emerged that HSCs possess a remarkable capacity to contribute to different types of tissue. Therefore, the ability to control or introduce the plasticity of cells has potentially many valuable applications. For example, functional hepatic reconstruction of FAH tyrosinase deficiency was achieved by using highly purified HSCs. Hepatocytes are derived from endoderm, thus it appears that mesodermally derived HSCs have a capacity to convert to an endodermal derivative (Lagasse et al, Nature Med. 6: 1229-1234, 2000). Another set of experiments points to even greater plasticity of HSCs, whereby a single stem cell derived from bone marrow demonstrated low level contribution to various epithelial tissues in many organs even though no data on functional capacity of these cells were presented (Krause et al, Cell 105: 369-377, 2001). Recent experiments with adult stem cells derived from enriched bone marrow populations further indicated a capacity to restore muscle cells after myocardial infarction (Orlic et al, Ann. NY Acad. Sci. 938: 221-230, 2001; Orlic et al, Nature 410: 701-705, 2001; Jackson et al, J. Clin. Invest. 107: 1395-1402, 2001).
The studies detailed above, clearly indicate that understanding and being able to introduce plasticity into cells has potentially valuable applications in for example tissue regeneration.
There is also increasing evidence that hematopoietic cells can retro- or de-differentiate into an earlier progenitor state. For example, chicken myelomonocytic cells transformed by a temperature sensitive (ts) mutant of ν-myb exhibit an immature phenotype, resembling myeloblasts at the permissive temperature. However, at a non-permissive temperature they shift into adherent, phagocytic, macrophage-like cells and cease dividing. Time-lapse experiments showed that this process was reversible with most adherent cells acquiring back blast morphology and re-entering the cell cycle within 2 to 3 days after shift to the permissive temperature (Beug et al, Genes Dev. 1: 277-286, 1987).
As yet, the majority of studies investigating trans-differentiation have been done with non-human cells as no reproducible and stable model exists which allows the generation of functionally differentiated myeloid or lymphoid cells from a single cell population by manipulation of the environment.
At present, all trans-differentiation studies within a hematopoietic lineage have been carried out in non-human models requiring transplantation into living host. Moreover, none of these model systems have demonstrated a high frequency of cells that switch to different lineages, thus limiting the efficacy of in vitro and in vivo studies. Most significantly, these models frequently involve the use of cell phenotypes which do not exist naturally, such as cells isolated from transgenic animals or artificially transformed cells.
There is limited or no data available in relation to transdifferentiation of mature effector cells. This results from a lack of a cell system or animal model which includes markers of mature phenotypes of B cells, T cells and myeloid cells, specifically markers such as CD19, CD4 or CD3, and CD15. The majority of experiments with B lineage transdifferentiation involve pre-B cells with very low frequency of conversion to mature CD19 positive cells. Whilst conversion between B lineage and myeloid and T lymphoid to myeloid has been achieved with limited success, no inter-conversion between B and T-lymphoid lineage has been observed.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.